Chromatography of 33 gibberellins on a gradient eluted silica gel partition column

Durley, Richard C.; Crozier, Alan; Pharis, R. P.; McLaughlin, G.E.

doi:10.1016/0031-9422(72)80098-0

Cited by 112 publications

(50 citation statements)

References 16 publications

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“…Plants were grown in a cooled greenhouse (21.5 C day, 15.5 C night) under natural photoperiod from September to December, 1973. Plant parts sampled are given in Table I. Batches of plant tissue, as described in Table I, were extracted in cold (<0 C) 80% methanol (10 g methanol to 1 g tissue) for GAs as described previously (3). Purification of the acidic, ethyl acetate-soluble fraction on Polyclar AT (PVP), an insoluble form of poly-N-vinylpyrrolidone (11), and charcoal-Celite columns (26) was followed by chromatography on Woelm silica gel partition columns (5,20) which were gradient-eluted using a four-chamber Varigrad system, chambers 1 to 4 being 50%, 65%, 85%, and 100% ethyl acetate in hexane (w/w), respectively. The 26 fractions collected were diluted serially over a wide range (1/150 to 1/3600 depending upon the dry weight and biological activity present) and bioassayed for GA-like activity in 0.5 al of 95% ethanol at each diluiton on 10 dwarf rice cv.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Native Gibberellins in the Internode, Nodes, Leaves, and Inflorescence of Developing Avena Plants

et al. 1976

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The native gibbereilins (GAs) of various organs of the Avena plant were analyzed by bioassay and gas chromatography-mass spectrometry (GC-MS) after silicic acid partition column chromatography. The major GA of the inflorescence was identified as GA3 by GC-MS, and this GA also forms the major component of the nodes, p-i internode, and roots as determined by GLC or chromatography/bioassay. The inflorescence and nodes are the major sources of native GAs, the last two leaves, internode, and roots having dgnificantly lower amounts of GA-like substances. In the internode, less polar GAs predominated at the lag stage of development, whereas by the log and plateau stages, the more polar GAs increased significantly.Since less polar GAs are early in oxidative interconversion sequences, this fnding indicates sequential conversion to more polar and probably more active GAs, during log phase growth of the p-i internode.Excised stem segments of Avena plants are known to be very sensitive in their growth response to exogenously supplied gibberellins (GAs), especially GA, and GA3 (13,15). In contrast, they show no significant growth promotion by other plant hormones such as IAA, kinetin, and ABA (14). In light of this marked specificity of oat stem segments to GAs, especially the more oxidized GA, and GA3, it is germane to ask the following questions: Where in the Avena plant are the primary sources of native GAs that may be regulating internodal elongation in situ? Are these GAs produced mainly in the internodes, or are they derived from other parts of the plant? What are the predominant forms (i.e. degree of oxidation) of GAs found in the Avena plant during rapid internode elongation?Past studies on native GAs in other cereals are relevant to the present investigation. Radley and co-workers (12, 21, 22) have characterized GA1-and GA3-like substances in barley by means of TLC, fluorimetry, and bioassay. Faull et al. (9) reached similar conclusions for barley GAs using paper, TLC, GLC, paper electrophoresis, and bioassay. Murphy and Briggs (18) identified GA3 and GA7 in germinating barley by novel means using crystallization to constant specific radioactivity with unlabeled carrier GA methyl ester after initial methylation of the endogenous GA with '4C-diazomethane. By techniques they estimated GA3 levels to be 1.5 ng/grain, a level which correlates well with bioassay estimates. Faull et al. (10) showed that 11-day-old barley seedlings synthesized an A,-like GA de novo from 14C02, and that the labeled GA was metabolized within 12 hr, implying a high rate of turnover of GAs in the seedlings. Nicholls (19) found very high levels (15 to 48 Ag/g, dry weight) of GAs in barley inflorescences.In the present study native GAs of Avena nodes, internodes (at 3 stages of elongation), roots, leaves, and inflorescences are analyzed by silicic acid partition column chromatography, being quantitated by bioassay, and in certain cases, GLC. Identification of one GA was made by GC-MS.4 These analyses have allowed the questions pose...

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Section: Methodsmentioning

confidence: 99%

Analysis of Native Gibberellins in the Internode, Nodes, Leaves, and Inflorescence of Developing Avena Plants

et al. 1976

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“…The biologically active GA1 (1) may be produced through C-3 hydroxylation, or alternatively, C-2 hydroxylation may yield the biologically inactive GA29. Given (5,15,19). Forty 10-ml fractions were collected rather than larger fractions previously used.…”

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confidence: 99%

Metabolism of Tritiated Gibberellin A₂₀ in Maize

Rood¹,

Koshioka²,

Douglas³

et al. 1982

Plant Physiol.

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After the application of 2.36 Curies per milUmole 12,3-'Hlgibbereuln A20 (GA20) to 21-day-old maize (Zea mays L., hybrid CM7 x CM49) plants, etiolated maize seedlings, or maturing maize cobs, a number of 3H-metabolites were observed. The principal acidic (pH 3.0), ethyl acetate-soluble metabolite was identified as I'HIGA1 on the basis of co-chromatography with standard I3HIGA1 on SiO2 partition, high resolution isocratic elution reverse phase C1i high performance liquid chromatography and gas-liquid chromatography radiocounting. Two other acidic metaboUtes were identified similarly as 13HIGA8 and C/D ring-rearranged I3HIGA2o, although gas-liquid chromatography radiocounting was not performed on these metabolites. Numerous acidic, butanol-soluble (e.g. ethyl acetate-insoluble) metabolites were observed with retention times on C1i high performance liquid chromatography radiocounting similar to those of authentic glucosyl conjugates of GA1 and GAs, or with retention times where conjugates of GA20 would be expected to elute. Conversion to I3HIGAi was greatest (23% of methanol extractable radioactivity) in 21-day-old maize plants. In etiolated maize seedigs, the C/D ring-rearranged I3HIGA2,-like metabolite was the major acidic product, while conversion to I3HIGA1 was low.Hedden et al. (10) have recently characterized GAui,5 GA44, GA17, GAi0, and GA20 from immature maize tassels by GLC-MS.Further, data from selected ion current monitoring GLC-MS indicate that GA1, GA29, and GA8 are probably also native in maize (10). These GAs are members of the probable early 13-hydroxylation pathway, a pathway which begins with GA12-aldehyde. The 13-hydroxylation pathway also exists in Phaseolus vulgaris (9) where GA20 is apparently a precursor of either GA1 or GA29 (19). In other plant systems, two principal metabolites have been tentatively identified following the application of [3HJGA20: 13,16). Since both GA1 and GA29 appear to be native in maize (10), a branch point in the metabolic pathway in maize may exist after GA20. The biologically active GA1 (1) may be produced through C-3 hydroxylation, or alternatively, C-2 hydroxylation may yield the biologically inactive GA29. Given 21-d-Old Maize Plants. Three kernels of the early maturing maize (Zea mays L.) hybrid CM7 x CM49 (17) were planted in each of 12 plastic pots (13 x 20 cm) filled with a mixture of peat moss and sand. Pots were placed in a growth room at 25/15°C (day/night), 14-h photoperiod (300 ,AE m-2 S-1). After emergence, plants were thinned to one per pot. Twenty-one days after planting (at which time the plants were still in the vegetative growth phase), 0.22 ,uCi [2,3-3HJGA20 (2.36 Ci/mmol) in 0.4 ml 60% aqueous ethanol was pipetted into the leaf whorl. Twenty-four, 48, 96, and 144 h after the addition of [3H]GA2o, three plant shoots were excised at the soil surface and 4 cm above the surface, yielding shoot cylinders which contained the apical meristems. These were rinsed with H20 and then homogenized separately at -40°C in MeOH:H20 (80:20, v/v

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“…These procedures significantly reduced the dry weight of the acidic fraction. Gibberellin-like substances in the sample were separated on a silica gel partition column (8,37) (8). The more polar acidic GAs such as GA32 and GA39 would not be "seen" in our study should they be present since they can only be extracted from the acidic, aqueous phase with butanol.…”

Section: Methodsmentioning

confidence: 99%

Endogenous Gibberellins of Pine Pollen

Kamieńska

Pharis²

1975

Plant Physiol.

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Thie endogenous gibberellins (GAs) of pollen of Pinus attenuata, P. coulteri, and P. ponderosa were bioassayed at hour 0, 3, 13, 24, 48 and 72 of germination. Dormant pollen showed relatively high GA activity throughout the elution spectrum (i.e. ranging from relatively nonpolar to highly polar). The maximum GA activity was obtained at hour 13 in more polar regions and especially in the zone corresponding to GA3 (for P. attenuata estimated as 230 micrograms of GA3/kilogram pollen). It is probable that the "nonpolar"GAs present in high quantities in dormant pollen and in early stages of germination were converted to "more polar"GAs as germination progressed. The amount of all GAs decreased after hour 13 of germination and by hour 72 no GAs could be detected. Among the species tested P. attenuata showed the highest over-all GA activity.

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Chromatography of 33 gibberellins on a gradient eluted silica gel partition column

Cited by 112 publications

References 16 publications

Analysis of Native Gibberellins in the Internode, Nodes, Leaves, and Inflorescence of Developing Avena Plants

Analysis of Native Gibberellins in the Internode, Nodes, Leaves, and Inflorescence of Developing Avena Plants

Metabolism of Tritiated Gibberellin A₂₀ in Maize

Endogenous Gibberellins of Pine Pollen

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Chromatography of 33 gibberellins on a gradient eluted silica gel partition column

Cited by 112 publications

References 16 publications

Analysis of Native Gibberellins in the Internode, Nodes, Leaves, and Inflorescence of Developing Avena Plants

Analysis of Native Gibberellins in the Internode, Nodes, Leaves, and Inflorescence of Developing Avena Plants

Metabolism of Tritiated Gibberellin A20 in Maize

Endogenous Gibberellins of Pine Pollen

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Metabolism of Tritiated Gibberellin A₂₀ in Maize